Project Summary/Abstract: Innate immunity?s importance in lung defense against pathogens has been heightened by the recent discovery of distinct intracellular pathogen recognition receptors that detect pathogen access to the cytosol of macrophages. These receptors include NOD-like receptors (NLRs) as well as NOD independent sensors such as pyrin. A critical function of the intracellular sensors is their direct regulation of the enzyme caspase-1 through a protein regulatory complex called an inflammasome. In an inflammasome, NLRs and NOD independent sensors interact with an adaptor protein ASC via pyrin domains (PYD) or caspase recruitment domains (CARD). This interaction induces caspase-1 dimerization and auto-activation. Caspase-1 then activates the precursors of IL-1? and IL-18, and active caspase-1 also drives a form of cell death of macrophages and lymphocytes known as pyroptosis. These events have been strongly associated with inflammatory lung disorders like pulmonary fibrosis. However, despite the conceptual advances provided by the inflammasome discovery, how this complex is assembled and controlled remains obscure, limiting our understanding of lung inflammation and our ability to create new therapies. In this context, the present application seeks to expand upon the inflammasome hypothesis by linking its structure and function to rapid events that involve tyrosine kinase activities. Our preliminary data point to a role for pyrin as a critical component of inflammasomes. We show that pyrin has the capacity to bring tyrosine kinases to the inflammasome complex to modulate ASC?s function via its phosphorylation. We therefore hypothesize that these pyrin-affiliated activities can promote interactions between ASC and caspase-1 via phosphorylations within conserved tyrosines in the CARD domains of ASC. Furthermore, we have found that resident alveolar macrophages differ from inflammatory macrophages in accordance with their relative expressions of pyrin. Utilizing these key discoveries, the project proposes the following specific aims, 1) to dissect the role of phosphorylation in ASC inflammasome regulation; 2) to determine pyrin?s role in modulating ASC phosphorylation dependent human inflammasome activation; and 3) to uncover critical pathways regulating caspase-1 activation by comparing resident human alveolar macrophage and macrophages from patients with idiopathic pulmonary fibrosis (IPF) for differences in pyrin and ASC dependent inflammasome signaling events. The successful completion of this project shows promise to provide new treatment targets for inflammatory lung disorders.